Biochemical saturation of molecules and its use

ABSTRACT

Provided herein are methods and compositions for selective enzyme-based hydrogenation of molecules as an alternative for current chemical catalyst-based methods. These methods include different enzymes and their related processes followed to obtain fully saturated or partially saturated molecules, without producing unwanted stereoisomers, for example trans-fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of PCT InternationalApplication Number PCT/US2021/071870, filed Oct. 14, 2021, designatingthe United States of America and published in the English language,which is an International Application of and claims the benefit ofpriority to U.S. Provisional Application No. 63/092,712, filed Oct. 16,2020, the disclosures of which are hereby expressly incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitledGEAE007C1, created Apr. 10, 2023, which is 81 KB in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates to the field of bioengineering andbiomanufacturing. More precisely, it relates to enzyme compositions andmethods of using the compositions for the production of partial or fullhydrogen saturated products of interest for food, cosmetic, orpharmaceutical use, among others.

Catalytic hydrogenation reactions have become key processes in variedmanufacturing industries, including agricultural, food, pharmaceutical,and fine chemicals, and the continuous innovations in hydrogenationprocesses highlight the importance of this technology (Machado, CurrOpinion Drug Disc Dev, 4(6), 745-755, 2001). Part of the continuouschallenges in catalytic hydrogenations is the ability to maximize theactivity while also maintaining high selectivity and reducingisomerization of the obtained products (Machado, (2001) Curr OpinionDrug Disc Dev, 4(6), 745-755; Patterson, (2011) Hydrogenation of Fatsand Oils, pp. 33-48, AOCS Press). This has become extremely importantfor applications in the food industry, in which unwanted isomers oflipid hydrogenation can have important health risks if consumed(Gebauer, (2013) Encyclopedia of Human Nutrition, pp. 288-292, 3rd ed.Academic Press).

Lipids are essential biological macromolecules, the main constituents offats and oils, and compounds of great commercial value, either ascommodities or as raw materials (Baumann, (1988) Angewandte ChemieInternational Edition, 27(1), 41-62). The melting point of a lipid isthe temperature at which it transitions from solid to liquid state.Lipids with low melting points are commonly liquid at moderate ambienttemperatures (around 25° C.). On the contrary, lipids with high meltingpoints are commonly semi-solid or solid at ambient temperature. Thisdistinction makes different lipids appropriate for differentmanufacturing uses. The melting point of lipids depends on a largedegree on the type of chemical bonding that occurs within the lipidhydrocarbon chain. Lipids that only contain single bonds between theircarbon atoms have high melting points and are commonly referred to assaturated because single bonded carbons are saturated with hydrogenatoms (Patterson, (2011) Hydrogenation of fats and oils, pp. 1-32, AOCSPress). Other lipids can contain one or more double bonds within thehydrocarbon chain and are thus unsaturated, and have low melting points.An important market need exists for lipid products that melt between 30°to 40° C. because products with these qualities are solid at roomtemperature but liquid when in contact with the skin or mouth. Thisproves as a key quality that impacts the texture of products, a criticalproperty for products of the food and cosmetic industries, and aproperty that is highly associated with product quality by consumers(Arellano, (2015) Specialty oils and fats in food and nutrition, pp.241-270, Woodhead Publishing).

Since the beginning of the 1900s, several chemical methods have beendevised that allow the chemical hydrogenation of vegetable oils, rich inunsaturated fatty acids, to generate saturated hardened fats. Theseprocesses are based on the reaction between molecular hydrogen and oilsin the presence of a metallic catalyst (like palladium or nickel), andhave been used to produce large yields of saturated lipids (Arellano,(2015) Specialty oils and fats in food and nutrition, pp. 241-270,Woodhead Publishing). Moreover, the same method can be applied tohydrogenate non-lipid hydrocarbon molecules, which generates products ofinterest for a variety of chemical industries (Baumann, (1988)Angewandte Chemie International Edition, 27(1), 41-62). One importantdrawback of chemical saturation of fatty acids for the food and cosmeticindustry uses, is that when the process is incomplete, it can yield highamounts of monounsaturated fatty acids with a trans configuration, ageometric configuration of the carbon double bond that occurs in a lowproportion in nature in comparison with the natural occurring cisconfiguration. Trans fats side products have different chemical andphysical properties than their natural counterparts due to theirchemical bond geometry, and their consumption has been linked withcardiovascular disease as well as other significant pathologies, leadingseveral countries to impose legal trans-fat limits (Gebauer, (2013)Encyclopedia of Human Nutrition, pp. 288-292, 3rd ed. Academic Press).

As a consequence of the reduction in demand for chemically hardenedvegetable oils, there has been an increase in the demand from theindustry for high melting point vegetable oils and butters, includingcocoa butter, shea butter and palm oil, which have the desirablehardness at room temperature and melting properties to replacechemically hardened oils. However, these alternatives are eitherexpensive, not sustainable, and face scrutiny due to their sourcing orenvironmental concerns due to promotion of deforestation (Meijaard,(2020) Nature plants, 6(12), pp. 1418-1426; Clough, (2009) Conservationletters, 2(5), 197-205; Elias, (2013). Sociologia Ruralis, 53(2),158-179). Due at least to these considerations, there is a dire need foralternative technologies that allow the controlled modification of thehydrogenation state of lipids, to generate the saturation of fatty acidswithout the occurrence of trans fatty acids, and to obtain greener andsafer methods to manipulate lipids and their melting temperatures.

SUMMARY

Some embodiments provided herein relate to methods that allow thehydrogenation of target molecules by means of an enzyme catalyst,through a novel repertoire of enzymes that allow modification ofsubstrates in order to obtain products that currently can only beobtained through chemical modification, or to obtain novel products notcurrently available in the market. Moreover, some embodiments providedherein relate to products and methods of making these products, whichhave specific spatial conformations while avoiding unwanted sideproducts (stereoselectivity), in comparison to other available chemicalhydrogenation methods that generate products with mixed spatialconformation products (mixes of left- and right-handed enantiomers).

Accordingly, provided herein are methods of saturating an unsaturatedmolecule. In some embodiments, the methods include contacting anunsaturated molecule with an enzyme to produce a saturated molecule, andrecovering the saturated molecule.

In some embodiments, the saturated molecule is fully or partiallysaturated. In some embodiments, the unsaturated molecule comprises anunsaturated alkene. In some embodiments, the unsaturated molecule is anunsaturated triglyceride or a free fatty acid. In some embodiments, theunsaturated molecule is vegetable oil. In some embodiments, theunsaturated molecule is olive oil or canola oil.

In some embodiments, contacting the unsaturated molecule with the enzymeis performed in a solvent. In some embodiments, contacting is performedfor a sufficient period of time to allow at least partial saturation.

In some embodiments, the enzyme is in solution. In some embodiments, theenzyme is immobilized. In some embodiments, the enzyme is immobilized ona polymeric support. In some embodiments, the polymeric support is aninsoluble polymer microbead.

In some embodiments, the enzyme is prepared by protein fermentation orchemical synthesis. In some embodiments, the enzyme is a purifiedenzyme. In some embodiments, the enzyme is a recombinant nickel bindingenzyme. In some embodiments, the enzyme has an amino acid sequence asset forth in SEQ ID NOs: 1-52, or having a sequence identity of at least75% to any one of SEQ ID NOs: 1-52. In some embodiments, the enzyme hasan amino acid sequence as set forth in SEQ ID NO: 15 or 40, or having asequence identity of at least 75% to any one of SEQ ID NOs: 15 or 40. Insome embodiments, the enzyme has a sequence having 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to anyone of SEQ ID NOs: 1-52. In some embodiments, the enzyme includes aconsensus sequence as set forth in SEQ ID NO: 53.

In some embodiments, the enzyme is a novel designed protein havinghydrogenase activity and comprising a substrate specific binding site.In some embodiments, the substrate specific binding site comprises oneor more alkene unsaturation sites. In some embodiments, the enzyme is ahydrogenase enzyme engineered to bind a non-canonical substrate. In someembodiments, the non-canonical substrate comprises one or more alkeneunsaturation sites. In some embodiments, the hydrogenase enzymecomprises a modified hydrophobic portion that supports the recognitionof an unsaturated acyl chain. In some embodiments, the enzyme is adehydrogenase enzyme engineered to bind a non-canonical substrate. Insome embodiments, the non-canonical substrate comprises one or morealkene unsaturation sites. In some embodiments, the enzyme is adesaturase enzyme. In some embodiments, the desaturase enzyme isengineered. In some embodiments, the desaturase enzyme is engineered tobind a transition metal in its active site. In some embodiments, theactive site comprises at least 2 cysteine residues that supporttransition metal binding. In some embodiments, the transition metal isnickel, iron, or palladium. In some embodiments, the active sitecomprises an arginine residue that is configured to support a frustratedLewis pair reaction.

Some embodiments relate to the following enumerated alternatives:

-   -   1. A method of saturating an unsaturated molecule, the method        comprising: contacting an unsaturated molecule with an enzyme to        produce a saturated molecule; and recovering the saturated        molecule.    -   2. The method of alternative 1, wherein the saturated molecule        is fully or partially saturated.    -   3. The method of any one of alternatives 1-2, wherein the        unsaturated molecule comprises an unsaturated alkene.    -   4. The method of any one of alternatives 1-3, wherein the        unsaturated molecule is an unsaturated triglyceride or a free        fatty acid.    -   5. The method of any one of alternatives 1-4, wherein the        unsaturated molecule is vegetable oil.    -   6. The method of any one of alternatives 1-5, wherein the        unsaturated molecule is olive oil or canola oil.    -   7. The method of any one of alternatives 1-6, wherein contacting        the unsaturated molecule with the enzyme is performed in a        solvent.    -   8. The method of any one of alternatives 1-7, wherein the        contacting is performed for a sufficient period of time to allow        at least partial saturation.    -   9. The method of any one of alternatives 1-8, wherein the enzyme        is in solution, or wherein the enzyme is immobilized.    -   10. The method of alternative 9, wherein the enzyme is        immobilized on a polymeric support.    -   11. The method of alternative 10, wherein the polymeric support        is a polymer microbead.    -   12. The method of any one of alternatives 1-11, wherein the        enzyme is prepared by protein fermentation or chemical        synthesis.    -   13. The method of any one of alternatives 1-12, wherein the        enzyme is a purified enzyme.    -   14. The method of any one of alternatives 1-13, wherein the        enzyme is a recombinant nickel binding enzyme.    -   15. The method of any one of alternatives 1-14, wherein the        enzyme comprises a consensus sequence as set forth in SEQ ID NO:        53.    -   16. The method of any one of alternatives 1-15, wherein the        enzyme has an amino acid sequence as set forth in SEQ ID NOs:        1-52, or having a sequence identity of at least 75% to any one        of SEQ ID NOs: 1-52, such as 75%, 80%, 85%, 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, or 99% sequence identity.    -   17. The method of any one of alternatives 1-16, wherein the        enzyme has an amino acid sequence as set forth in SEQ ID NO: 15        or 40, or having a sequence identity of at least 75% to any one        of SEQ ID NOs: 15 or 40, such as 75%, 80%, 85%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.    -   18. The method of any one of alternatives 1-17, wherein the        enzyme is a novel designed protein having hydrogenase activity        and comprising a substrate specific binding site.    -   19. The method of alternative 18, wherein said substrate        specific binding site comprises one or more alkene unsaturation        sites.    -   20. The method of any one of alternatives 1-19, wherein said        enzyme is a hydrogenase enzyme engineered to bind a        non-canonical substrate.    -   21. The method of alternative 20, wherein said non-canonical        substrate comprises one or more alkene unsaturation sites.    -   22. The method of any one of alternatives 20-21, wherein the        hydrogenase enzyme comprises a modified hydrophobic portion that        supports the recognition of an unsaturated acyl chain.    -   23. The method of any one of alternatives 1-22, wherein said        enzyme is a dehydrogenase enzyme engineered to bind a        non-canonical substrate.    -   24. The method of alternative 23, wherein said non-canonical        substrate comprises one or more alkene unsaturation sites.    -   25. The method of any one of alternatives 1-24, wherein said        enzyme is a desaturase enzyme.    -   26. The method of alternative 25, wherein said desaturase enzyme        is engineered.    -   27. The method of any one of alternatives 25-26, wherein said        desaturase enzyme is engineered to bind a transition metal in        its active site.    -   28. The method of alternative 27, wherein said active site        comprises at least 2 cysteine residues that support transition        metal binding.    -   29. The method of any one of alternatives 27-28, wherein the        transition metal is nickel, iron, or palladium.    -   30. The method of any one of alternatives 27-29, wherein said        active site comprises an arginine residue that is configured to        support a frustrated Lewis pair reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the embodiments described herein can beobtained, a more particular description will be rendered by reference tospecific embodiments thereof which are illustrated in the appendeddrawings. Understanding that these drawings depict only exemplaryembodiments and are not therefore to be considered to be limiting of itsscope, the embodiments herein will be described and explained withadditional specificity and detail through the use of the accompanyingdrawings in which:

FIG. 1 depicts an exemplary method for enzymatic biochemical saturationas described in some embodiments herein.

FIG. 2 depicts a ribbon model (left) and a space filled Van der Waalssurface model (right) of an example engineered desaturase enzyme. Theimages depict the molecular docking of tristearin in the active site ofdesaturase 9, and include the metal atoms coordinated in the activesite.

FIG. 3 depicts an exemplary mechanism for H₂ oxidation of fatty aciddouble bonds by a nickel binding active site. Hydrogenase-like reactionswill have the potential to reversibly convert molecular hydrogen intoprotons and electrons. Furthermore, and given the specific conditions,free protons will have the potential to form hydride ions with the ionmetals on site, later promoting the oxidation and consequent reductionof a double carbon bond. For example, when the total number of carbonsin the fatty acid is 18, this would represent the conversion of oleicacid into stearic acid.

FIG. 4 depicts a frustrated Lewis pair reaction. In order to improve theoxidation of H₂, a frustrated Lewis pair reaction can be developed inthe active site of a desaturase, where the reverse reaction of thisenzyme can consequently be performed.

FIG. 5 depicts an exemplary enzyme and active site for alkenehydrogenation. The large subunit of a hydrogenase is shown on the left,shaded by the corresponding hydrophobicity level for each residue(white=less hydrophobic, dark=more hydrophobic). Alkene hydrogenationcan occur if the circled region is modified for more hydrophobicresidues (right). The recognized alkene can be enzymaticallyhydrogenated by the catalytic core, with the participation of thecorresponding metal nickel-iron core, and arginine 509 (R509).

FIG. 6 depicts an example of enzymatic hydrogenation of a fatty acid,showing a schematic representation of an example mechanism of fatty acidhydrogenation by a modified hydrogenase. The enzymatic reaction startsby the incorporation of molecular hydrogen to the active site, where itis split in two protons by the [Ni—Fe] metal core and an oppositearginine. Each proton can bind to the carbon-carbon double bond sectionof an oleic acid by proximity, forming stearic acid. The latter isremoved and replaced by another oleic acid, and a new reaction starts.

FIG. 7A depicts a schematic representation of a novel enzyme design.FIG. 7B depicts nickel binding properties as defined by fluorescenceassay. Nickel binding in a designed pocket greatly reduces the intrinsicprotein fluorescence in the protein ID #8 (having an amino acid sequenceas set forth in SEQ ID NO: 8) novel enzyme design, but not to the samedegree in a design lacking the nickel binding domain (protein ID #6;having an amino acid sequence as set forth in SEQ ID NO: 6).

FIGS. 8A and 8B depict results of desaturation of an engineereddesaturase enzyme. FIG. 8A depicts a canonical desaturation of stearicacid as reflected by the increase in the iodine value. FIG. 8B depicts anon-canonical reaction, showing variation of the iodine value of oliveoil after five hours of incubation with the desaturase enzyme andhydrogen gas.

FIG. 9 depicts the texture variation of olive oil after treatment withthe enzyme described in FIGS. 8A and 8B. The upper fraction of thetreated oil (right) presents a spreadable texture.

FIGS. 10A and 10B depict results of enzymatic saturation of free fattyacids with an exemplary enzyme as described herein. Native and novelengineered enzymes were expressed and purified from E. coli and used tosaturate fatty acids. FIG. 10A depicts expression of Protein IDs #6-8(SEQ ID NOs: 6-8) as detected from lysates of E. coli by SDS-PAGE.Asterisks show bands corresponding to proteins of interest. FIG. 10Bdepicts enzymatic saturation of free fatty acid oil comprised of 90%oleic acid. The oil was saturated using Protein ID #8 (SEQ ID NO: 8)incubated with NiCl₂ and with H₂ bubbling for 8 hours. As a control thesame reaction was run with NiCl₂ but without Protein ID #8. The fattyacid composition was analyzed by GC-FID and compared to the untreatedoil.

FIGS. 11A-11C depict results of enzymatic hydrogenation of canola oilusing metal binding proteins as catalysts. Native and engineered metalbinding proteins were expressed and purified from E. coli and used tosaturate fatty acids. FIG. 11A depicts expression of Protein ID #40 (SEQID NO: 40) and FIG. 11B depicts expression of Protein IDs #41-42 (SEQ IDNOs: 41 and 42) as detected from lysates of E. coli by SDS-PAGE.Asterisks show bands corresponding to proteins of interest. FIG. 11Cdepicts enzymatic saturation of canola oil using Protein ID #40incubated with NiCl₂ and with H₂ bubbling for 8 hours. The fatty acidcomposition was analyzed by GC-FID and compared to the untreated oil.

FIGS. 12A and 12B depict a molecular model representation of a designedmetal binding site. FIG. 12A shows protein residues involved in theinteraction are labeled, such as histidine, aspartic acid, and glutamicacid. FIG. 12B shows octahedral coordination sites.

FIGS. 13A and 13B depict enzymatic hydrogenation of canola oil usingnovel protein as catalyst. A native enzyme (Prot.ID #13-SEQ ID NO: 13)was modified to improve it affinity towards free fatty acids, and tocontain a metal binding domain (Prot.ID #15-SEQ ID NO: 15), and used topartially saturate canola oil FIG. 13A shows expression of Protein IDs#15 (SEQ ID NO: 15) and #13 (SEQ ID NO: 13) as detected from lysates ofE. coli by SDS-PAGE. Asterisks show proteins of interest. S: solublefraction, I: Insoluble fraction. FIG. 13B shows enzymatic saturation ofcanola oil using Protein ID #15 (SEQ ID NO: 15) incubated with NiCl₂ andwith H₂ bubbling for 8 hours. The fatty acid composition was analyzed byGC-FID and compared to the untreated oil.

DETAILED DESCRIPTION

Provided herein are methods and processes for the hydrogenation oftarget molecules containing unique or multiple double or triple atomicbonds. In some embodiments, the methods use one or more proteincatalysts (also referred to herein as enzyme catalysts) in solublesuspension or immobilized form together with a hydrogen donor compoundto generate the saturation of the target double or triple bonds toobtain a partially or fully saturated product.

In the description that follows, the terms should be given their plainand ordinary meaning when read in light of the specification.

“About” as used herein when referring to a measurable value is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue.

As used herein, the term “hydrogenation” has its ordinary meaning asunderstood in light of the specification, and refers to a reductionreaction wherein hydrogen is the reducing agent. As used herein, theterm “saturation,” “saturated,” or “unsaturated” has its ordinarymeaning as understood in light of the specification, and refers to adegree of single bonds in a lipid or fatty acid chain. For example, asaturated fatty acid refers to a lipid or fatty acid chain having all orpredominantly all single bonds, whereas an unsaturated fatty acid refersto a lipid or fatty acid chain having at least a single double bond. Apartially saturated molecule refers to a molecule that has saturatedbonds and unsaturated bonds. For example, a partially saturated moleculemay include at least one single bond in a lipid or fatty acid.

As used herein, the term “hydrogenase activity” has its ordinary meaningas understood in light of the specification, and refers to an enzymehaving an ability to catalyze hydrogenation in a substrate. As usedherein, the term “substrate” has its ordinary meaning as understood inlight of the specification, and refers to a molecule that specificallybinds to a specific binding site in an enzyme, and upon which catalysistakes place.

As used herein the term “molecule” has its ordinary meaning asunderstood in light of the specification, and refers to a range ofcompounds including but not restricted to: unsaturated aliphatichydrocarbon compounds (alkenes, alkynes, alkyl cycloalkenes,cycloalkenes, cycloalkynes, dienes, polyenes, polyynes and theirderivatives); aromatic hydrocarbons; heterocyclic aromatic hydrocarbons,polycyclic aromatic hydrocarbons; double bonding organic molecules(aldehydes, amides, carboxylic acids, carboxylate esters, imines,ketones, thioketones, thiols), double bonding inorganic molecules (azocompounds, alkylidenesilanes, disulfurs, germenes, nitroso compounds,plumbenes, silenes, sulfoxides, sulfones, stannenes); triple bondingorganic molecules (alkynes, cyanides, isocyanides); mono- andpolyunsaturated lipids (including triglycerides, diglycerides,monoglycerides, free fatty acids, phospholipids, waxes, cholesterols andtheir esters and derivatives). As used herein, “molecule(s)” can referto the pure compound, or mixtures of these compounds as found inproducts of animal, vegetable, or synthetic origin.

Examples of unsaturated fatty acids include, for example, stearidonicacid, cervonic acid, myristoleic acid, palmitoleic acid, sapienic acid,oleic acid, elaidic acid, linoleic acid, linoelaidic acid,alpha-linolenic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,arachidonic acid, erucic acid, docosahexaenoic acid, docosatetraenoicacid, vaccenic acid, paullinic acid, gondoic acid, erucic acid, nervonicacid, mead acid, and eicosapentaenoic acid.

Examples of saturated fatty acids include, for example, propionic acid,butyric acid, valeric acid, caproid acid, enanthic acid, caprylic acid,pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylicacid, myristic acid, pentadecylic acid, palmitic acid, margaric acid,stearic acid, nonadecylic acid, arichidic acid, heneicosylic acid,behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid,cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissicacid, hentriacontylic acid, lacceroic acid, psyillic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid,octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid.

As used herein, the term “alkyl” refers to straight chained and branchedsaturated hydrocarbon groups. The term Cn means the alkyl group has “n”carbon atoms. For example, C₄ alkyl refers to an alkyl group that has 4carbon atoms. C₁₋₆ alkyl refers to an alkyl group having a number ofcarbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms),as well as all subgroups (e.g., 1-5, 2-5, 1-4, 2-5, 1, 2, 3, 4, 5, and 6carbon atoms). Unless otherwise indicated, an alkyl group can be anunsubstituted alkyl group or a substituted alkyl group.

As used herein, the term “alkene” is defined identically as “alkyl”except for containing at least one carbon-carbon double bond. Thus, analkene unsaturation refers to an unsaturated alkene molecule.

In some embodiments, the term “fat” or “fat molecule” refers to atriglyceride, diglyceride, or monoglyceride. The term also may refer toa free fatty acid, phospholipid, or a wax. The term “lipid” can refer tothe purified molecule, as well as mixtures of these molecules found infat products of animal, vegetal or synthetic origin, including oils,lards, butters and tallows.

“Hydrogen donor” has its ordinary meaning as understood in light of thespecification, and refers to any compounds that are able to release ahydrogen atom upon interacting with the protein catalysts, including butnot limited to hydrogen gas, nucleotidic coenzymes (NADH, NADPH, FADH₂)or donor molecules such as formic acid, isopropanol ordihydroanthracene.

The reaction may also contain, in addition to the protein catalyst,target molecule(s) and hydrogen donor, a suitable polar or nonpolarsolvent or mixture thereof. As used herein, the term “solvent” has itsordinary meaning as understood in light of the specification, and refersto a substance capable of at least partially dissolving anothersubstance. Solvents may be liquids at room temperature. Solvents may beorganic solvents (for example, having at least one carbon atom) andwater. In some embodiments, the solvent may be formed by the combinationof two or more organic solvents, or by the combination of an organicsolvent and water.

Suitable organic solvents may include, for example, optionallychlorinated aliphatic, cycloaliphatic or aromatic hydrocarbons such asn-pentane, n-heptane, n-octane, cyclopentane, cyclohexane, benzene,toluene, xylenes and chlorobenzene; aromatic, aliphatic and cyclicethers such as anisole, diethyl ether, di-isopropyl ether,tetrahydrofuran, methyltert-butyl ether and dioxane; N-substitutedmorpholines, such as N-methylmorpholine and N-formylmorpholine;nitriles, particularly benzonitrile and alkylnitriles having 2 to 5carbon atoms, such as propionitrile and butyronitrile;3-methoxypropionitrile and 3-ethoxypropionitrile; dialkyl sulfoxidessuch as dimethyl and diethyl sulfoxide; N,N-dialkylamides of aliphaticmonocarboxylic acids having 1 to 3 carbon atoms in the acid part, suchas N,N-dimethylformamide and N,N-dimethylacetamide; alcohols having upto 8 carbon atoms, such as ethanol, n-propanol and tert-butanol;aliphatic and cyclic ketones, such as acetone, diethyl ketone, methylisopropyl ketone, cyclopentanone, cyclohexanone,1,3-dimethyl-2-imidazolidinone and1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone; tetramethylurea;esters, such as esters of carbonic acid, such as diethyl carbonate;nitromethane; alkyl or alkoxyalkyl esters of aliphatic monocarboxylicacids having a total of 2 to 8 carbon atoms, such as methyl, ethyl,n-butyl and isobutyl acetate, ethyl and n-butyl butyrate, and1-acetoxy-2-ethoxyethane and 1-acetoxy-2-methoxyethane or triethylphosphate, or chlorinated aliphatic hydrocarbons, such asdichloromethane and chloroform, and diethyl ether, tert-butyl methylether, tetrahydrofuran, dioxane, ethanol, n-propanol, acetonitrile andtert-butanol.

The reaction can be performed in conditions that partially or totallyremove the presence of oxygen, including vacuum, displacement with othergases, or reaction with oxygen-consuming chemicals; in order to avoidthe oxidation of double or triple bonds and the formation of unwantedside-products.

The temperature and pressure conditions of the reaction can bemanipulated as well, in order to modulate conversion rates and to aswell favor or disfavor the production of specific reaction products.

In a first step of some embodiments of the reaction, an enzyme catalysthydrolyzes a hydrogen gas or a hydrogen donor molecule and thentransfers the free positively charged hydrogen to the double or tripleatomic bond of the substrate molecule, reducing the atoms forming thisbond.

As used herein, the term “purity,” “pure,” or “purified” has itsordinary meaning as understood in light of the specification and refersto physical separation of a substance of interest from other substances.In certain embodiments, the “purity” of any given agent (e.g., an enzymeor a saturated or unsaturated molecule) in a composition may bespecifically defined. For instance, certain compositions may include,for example, an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% pure, including all decimals in between, asmeasured, for example and by no means limiting, by biochemical oranalytical methods.

The term “isolated” is meant material that is substantially oressentially free from components that normally accompany it in itsnative state.

The term oil refers to a lipid composition, which can include theaforementioned molecules, in different proportions, where the meltingtemperature is lower than the ambient temperature, causing it to be in aliquid state, while the term fat, refers to compounds, which at roomtemperature are in a solid state.

An exemplary method for saturating an unsaturated molecule usingembodiments of the methods provided herein is summarized in FIG. 1 . Insome embodiments, the reaction may be carried out in a reactorcontaining a substrate lipid with one or more unsaturated molecules(carbon-carbon double bonds) in a suitable solvent mixed with hydrogengas or a hydrogen donor molecule (i.e., NADH, NADPH or FADH₂). Thereaction may also contain an enzyme catalyst in a suitable solvent.

In some embodiments, the methods are carried out as described in FIG. 1. In some embodiments, a first step includes providing a substrate as astarting material, which may include a molecule with one or multipledouble or triple bonds. In some embodiments, the starting material ispure or in mixed form. The substrate may then be subjected to anenzymatic saturation reaction, which includes contact with an enzymecatalyst as described and provided herein with a hydrogen donor. In someembodiments, the enzyme catalyst (also referred to herein as a proteincatalyst) is a native or engineered enzyme. In some embodiments, theenzyme catalyst is a desaturase, a hydrogenase, a reductase, a metalbinding protein, or a novel engineered enzyme. In some embodiments, thehydrogen donor is a hydrogen gas. In some embodiments, the enzymaticsaturation reaction includes transfer of a free positively chargedhydrogen to a carbon-carbon double bond of the substrate (startingmaterial), thereby producing a saturated carbon-carbon bond, resultingin a product. This step can be carried out at temperature and pressureconditions that can be higher or lower than normal room conditions, inorder to improve the catalytic efficiency of the process, whilepreserving the function of the enzyme.

In some embodiments, the reaction is carried out at a temperatureranging from about −10° C. to about 100° C., such as −10, −5, 0, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100° C., or at a temperature within a range defined by any two of theaforementioned values. In some embodiments, the reaction is carried outat a pressure ranging from about 0.5 atm to about 5 atm, such as 0.5, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 atm, or at a pressure within a rangedefined by any two of the aforementioned values. In some embodiments,the reaction is carried out for a time period ranging from about 0.1hours to about 24 hours, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or fora period of time within a range defined by any two of the aforementionedvalues.

In the second step of some embodiments, the product is separated,thereby recovering at least a partially saturated lipid. In someembodiments, the product includes selective obtention of cis partiallysaturated and/or fully saturated target molecules.

In some embodiments, the reaction may be carried out in a continuousflux reactor containing the substrate lipid in a suitable solvent mixedwith hydrogen or a hydrogen donor molecule and the enzyme catalyst maybe embedded in a solid-state polymer, glass matrix or any type ofimmobilization surface. In this case the enzyme can be reutilizedmultiple times.

In some embodiments, the enzyme catalyst is an engineered metal bindingprotein. In some embodiments, the enzyme catalyst contains a metalbinding site and/or domain, such as nickel, iron, palladium, or anyother transition metal. In some embodiments, the metal binding site iscoordinated in an octahedral or tetrahedral coordination geometry (asshown in FIG. 12B), with amino acids such as histidine, cysteine,glutamic acid, and/or aspartic acid, allowing the oxidation of molecularhydrogen (H₂) in order to reduce lipid molecules in a mixture (as shownin FIG. 12A).

In some embodiments, the metal binding site includes a nickel bindingmotif in the form of His-Xaa(4)-Asp-His (SEQ ID NO: 53). Table 1 depictsvarious engineered and natural enzymes that may be used in the methodsdescribed herein.

TABLE 1 SEQ ID NO Sequence 1 VISYDNYVTILDEETLKAWIAKLEKAPVFAFDTETDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAP DQISRERALELLKPLLEDEKALKVGQNLKYDRGILANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAE RWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIEMPL VPVLSRIER 2VISYDNYVTILDEETLKAWIAKAEKAPVFAFDTHT DSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGHELKHDRGIL ANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRHA AEHADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIER 3 VISYDNYVTILDEETLKAWIAKAEKAPVFAFDTCTCCCSNISALGCNVLCLIEPGVAAYIPVAHDYLDAP DQISRERALELLKPLLEDEKALKCHIELKLCRDGICNYGIELRGIAFCCIFESYILNSVAGRHCCCCLAE RWLKHKTITFEEIAGKGKNQLTFCQIALEEAGRHACDCARHTLQLHLKMWPDLQKHKGPLNVFENIEMPL VPVLSRIER 4VISYDNYVTILDEETLKAWIAKAEKAPVFAHDHHT DSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGHELKHDRGIL ANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRHA AEHADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIER 5 VISYDNYVTILDEETLKAWIAKAEKAPVFADCHCTFEQNNISAMKCCQAICIEPGVAAYIPVAHDYLDAP DQISRERALELLKPLLEDEKALKHCCELPFFRCCCMNYGIELRGIAFYCFCESYILNSVAGRHAWRCLAE RWLKHKTITFEEIAGKGKNQLTACQIALEEAGRHACCCAVYTLQLHLKMWPDLQKHKGPLNVFENIEMPL VPVLSRIER 6AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGL RVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQNQWAPDIQYYNGKYWLYYSVSSFGSNTSAIGLASST SISSGGWKDEGLVIRSTSSNNYNAIDPELTFDKDGNPWLAFGSFWSGIKLTKLDKSTMKPTGSLYSIAAR PNNGGALEAPTLTYQNGYYYLMVSFDKCCDGVNSTYKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGN DQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLINDLNWSSGWPSY 7 AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGLRVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQN QWRPDIQYYNGKYWLYYSVSSFGSNTSAIGLASSTSISSGGWKDEGLVIRSTSSNNYDAEDPELTFDKDG NPWLAFGDFRSGIKLTKLDKSTMKPTGSLYSIAARPNNGGALEAPTLTYQNGYYYLMVSFDKCCDGVNST YKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGNDQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLIN DLNWSSGWPSY 8AFWGASNELLHDPTMIKEGSSWYALMTGLTEERGL RVLKSSDAKNWTVQKSIFTTPLSWWSNYVPKYGQNQWRPDIQYYNGKYWLYYSVWSFGSNTSAIGLASST SISSGGWKDEGLVIYSTSSNNYDAEDDELTFDKDGNPWLAFGDFRSGIKLTKLDKSTMKPTGSLYSIAMR PNNGGALEAPTLTYQNGYYYLMVSFFKCCWGVNFTYKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGN DQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLINDLNWSSGWPSY 9 AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGLRVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQN QHAPDIQYYNGKYWLYYSVESFGSNTSAIGLASSTSISSGGWKDEGLVIRSTSSNNYHADHPELTFDKDG NPWLAFGHHESGIKLTKLDKSTMKPTGSLYSIAARPNNGGALHAPTLTYQNGYYYLMVSFDKCCDGVNST YKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGNDQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLIN DLNWSSGWPSY 10AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGL RVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQNQHAPDIQYYNGKYWLYYSVESCGCKCCVIGLASST SISSGGWKDEGLVIRSTSSNNYLACHPELTFDKDGNPWLAFGCHESCCCLTKLDKSTMKPTGSLYSIAAR PNNGGNSVACTLTYQNGYYYLMVSFDKCCDGVCCACKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGN DQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLINDLNWSSGWPSY 11 AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGLRVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQN QHAPDIQYYNGKYWLYYSVESFGSNTSAIGLASSTSISSGGWKDEGLVIRSTSSNNYHACHPELTFDKDG NPWLAFGCCESGIKLTKLDKSTMKPTGSLYSIAARPNNGGALDAPTLTYQNGYYYLMVSFEKCCDGVRST YKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGNDQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLIN DLNWSSGWPSY 12AFWGASNELLHDPTMIKEGSSWYALGTGLTEERGL RVLKSSDAKNWTVQKSIFTTPLSWWSNYVPNYGQNQHAPDIQYYNGKYWLYYSCRSYCCCSCCIGLASST SISSGGWKDEGLVIRSTSSNNYHACHPELTFDKDGNPWLAFGCCESCCCLTKLDKSTMKPTGSLYSIAAR PNNGCCCCACTLTYQNGYYYLMVSFEKCCDGVYSCCKIAYGRSKSITGPYLDKSGKSMLEGGGTILDSGN DQWKGPGGQDIVNGNILVRHAYDANDNGIPKLLINDLNWSSGWPSY 13 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVITGKEDVIVQGDKAIVLANGSPLLARVTGAGCLLGGIIAGFLFRETEPDIEALIEAVSVFN IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 14 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVIHGKEDVIVQGDKAIVLANGSPLLARVTGAGCLLGGIIAGFLFRETEPDIEALIEAESVHH IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 15 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKCIYNEILALIDDTATLDAVTIAKKA YAIYKTAIVPMGKEVVSWQGDKAIHFDPPMPSHFPVTGAGCLGMTISAGFLFRETEPDIEALDTFCSCCH IYGCEAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 16 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVIHGKEHVIVQGDKAIVLANGSPLEARVTGAGCLHGGIIAGFLFRETEPDIEALIEAHSVHH IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 17 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKAKDMEILALIDDTATLDAVTIAKKA YAIYKTAIVKYGCGCVSKQGDKAICCCEGRWKQARVTGCACDFGNLIAGFLFRETEPDIEALECCCSMWH IQCWEAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 18 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVICGKECVIVQGDKAIVLANGSPLEARVTGAGCLCGGIIAGFLFRETEPDIEALIEAHSVHC IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 19 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKVQCPEILALIDDTATLDAVTIAKKA YAIYKTAIVICGWSCVHQQGDKAIIVCKGHMTAWKVTGLWCLCGSMVCGFLFRETEPDIEALEGCISHIC ICVKKAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 20 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVIITEGDVIVQGDKAIVLANGSPLLARVTGAGCLHGGIIAGFLFRETEPDIEALIEACSVSE IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 21 MNYLNNIRIENPLTICYTNDVVKNFTANGLLSIGASPAMSEAPEEAEEFYKVAQALLINIGTLTAQNEQD IIAIAQTANEAGLPIVFDPVAVGASTYRKQFCKLLLKSAKVSVIKGNASEILALIDDTATLDAVTIAKKA YAIYKTAIVIIEEGDVIVQGDKAIVLANGSPLLARVTGAGCLHGGIIAGFLFRETEPDIEALIEACSVIE IAAEVAAENENCGGPGTFSPLLLDTLYHLNETTYQQRIRIQEVE 22 MSALTDLFPLPIVQAPMAGGVSVPQLAAAVCEAGGLGFLAAGYKTADGMYQEIKRLRGLTGRPFGVNVFM PQPELGAVEVYAHQLAGEAAWYETELGDPDGGRDDGYDAKLAVLLDDPVPVVSFHFGVPDREVIARLRRA GTLTLVTATTPEEARAVEAAGADAVIAQGVEAGGHQGTHRDSSEDDGAGIGLLSLLAQVREAVDIPVVAA GGIMRGGQIAAVLAAGADAAQLGTAFLATDESGAPGPHKRALTDPLFARTRLTRAFTGRPARSLVNRFLR EHGPYAPAAYPDVHHLTSPLRKAAAKAGDAQGMALWAGQGHRMARELPAGRLVEVLAAELAEARTALS 23 MSALTDLFPLPIVQHDHHGGVSVPQLAAAVCEAGGLGFLAAGYKTADGMYQEIKRLRGLTGRPFGVDVHM PQPELGAVEVYAHQLAGEAAWYETELGDPDGGRDDGYDAKLAVLLDDPVPVVSFHFGVPDREVIARLRRA GTLTLVTATTPEEARAVEAAGADAVIAQGVEAGGHQGTHRDSSEDDGAGIGLLSLLAQVREAVDIPVVAA GGIMRGGQIAAVLAAGADAARLGTAFLATDESGAPGPHKRALTDPLFARTRLTRAFTGRPARSLVNRFLR EHGPYAPAAYPDVHHLTSPLRKAAAKAGDAQGMALWAGQGHRMARELPAGRLVEVLAAELAEARTALS 24 MSALTDLFPLPIVQHDHHLGASVPQLAAAVCEAGGLGFLAARYKTADGMYQEIKRLRGLTGRFFGVDVHM PQPELGAVEVYAHQLAGEAAWYETELGDPDGGRDDGYDAKLAVLLDDPVPVVSFHFGVPDREVIARLRRA GTWTLFTATTPEEARAVEAAGADAVIAQGVTAGGHQGSHRDSSEDDGAGIGLLSLLAQVREAVDIPVVAA GGTMRGGQIAALLAAGFDAARLGTAFLATDESGAPGPHKRALTDPLFARTRLTRAFTGRPARSLVNRFLR EHGPYAPAAYPDVHHLTQPLRKAWYKAGDAQGMALWAGQGHRMARELPAGRLVEVLAAELAEARTALS 25 MQTFVHEAGRLPAHIAAELGSYRYRVFVEQLGWQLPSEDEKMERDQYDRDDTVYVLGRDANGEICGCARL LPTTRPYLLQEVFPHLLADEAPRSAHVWELSRFAATAWSVRPMLAAAVECAARRGARQLIGVTFCSMERM FRRIGVHAHRAGAPVSIDGRMVVACWIDIDAQTLAALDLDPALC 26 MQTFVHEAGRLPAHIAAELGSYRYRVFVEQLGRQLPSEDEKMERDQYDRDDTVYVLGRDANGEICGCARL LPTTRPYLLQEVEPHELADEAPRSAHVWELSRFAATAWSVRPMLAAAVECAARRGARQLIGHTDCSMERM FRRIGVHAHRAGAPVSEDGRMEVACWIDIDAQTLAALDLDPALC 27 MQTFVHEAGRLPAHIAAELGSYRYQNFCSAQGRQLPSEDEKMERDQYDRDDTVYVLGRDANGEICGCYSR YPTTRPKDEVEFEPHEFADEAPRSAHVFPVSIFAWTAWSVRPMLAAAVECAARRGARQLIGDTECRMERM FRRIGVHAHRAGAGVVEEYRTEFICWIDIDAQTLAALDLDPALC 28 MQPDPIAADILRKEPEQETFVRLNVPLEVPTSDEVEAYKCLQECLELRKRYVFQETVAPWEKEEPFAHYP QGKSDHCFEMQDGVVHVFANKDAKEDLFPVADATAFFTDLHHVLKVIAAGNIRTLCHRRLVLLEQKFNLH LMLNADKEFLAQKSAPHRDFYNVRKVDTHVHHSACRFLGEITKQVFSDLEASKYQMAEYRISIYGRKMSE WDQLASWIVNNDLYSENVVWLIQLPRLYNIYKDMGIVTSFQNILDNIFIPLFEATVDPDSHPQLHVFLKQ VVGFDLVDDESKPERRPTKHMPTPAQWTNAFNPAFSYYVYYCYANLYVLNKLRESKGMTTITLRPHSGEA GDIDHLAATFLTCHSIAHGINLRKSPVLQYLYYLAQIGLAMSPLSNNSLFLDYHRNPFPVFFLRGLNVSL STDDPLQIHLTKEPLVEEYSIAASVWKLSACDLCEIARNSVYQSGFSHALKSHWIGKDYYKRGPDGNDIH KTNVPHIRVEFRDTIWKEEMQQVYLGKAVISDEVEH 29 MQPDPIAADILRKEPEQETFVRLNVPLEVPTSDEVEAYKCLQECLELRKRYVFQETVAPWEKEEPFAHYP QGKSDHCFEMQDGVVHVFANKDAKEDLFPVADATAFFTDLHHVLKVIAAGNIRTLCHRRLVLLEQKFNLH LMLNADKEFLAQKSAPHRDFYNVRKVDTHVHHSACRFLGEITKQVFSDLEASKYQMAEYRISIYGRKMSE WDQLASWIVNNDLYSENVVWLIQLPRLYNIYKDMGIVTSFQNILDNIFIPLFEATVDPDSHPQLHVFLKQ VVGFDLVDDESKPERRPTKHMPTPAQWTNAFNPAFSYYVYYCYANLYVLNKLRESKGMTTITLRPHSGEA GDIDHLAATFLTCHSIAHGINLRKSPVLQYLYYLAEIGLAMSPLSNNSLFLDYHRNPFPVFFLRGLHVSL STDDPLQIHLTKEPLVEEYSIAASVWKLSACDLCEIARESVHHSGFSHALKSHWIGKDYYKRGPDGNDIH KTNVPHIRVEFRDTIWKEEMQQVYLGKAVISDEVEH 30 MQPDPIAADILRKEPEQETFVRLNVPLEVPTSDEVEAYKCLQECAELMKRYVFQETVAPWEKEEPFAHYP QGKSDHCFEMQDGVVHVFANKDAKEDLFPVADATAFFTDLHHVLKVIAAGNIRTLCHRRLVLLEQKFNLH LMLNADKEFLAQKSAPHRDFYNVRKVDTHVHHSACRFLGEITKQVFSDLEASKYQMAEYRISIYGRKMSE WDQLASWIVNNDLYSENVVWLIQLPRLYNIYKDMGIVTSFQNILDNIFIPLFEATVDPDSHPQLHVFLKQ VVGFDLVDDESKPERRPTKHMPTPAQWTNAFNPAFSYYVYYCYANLYVLNKLRESKGMTTITCRPHSGEA GDIDHLAATFLTCVSIAHGINLRKSPVLQYLYYLAEIFLAKSPLSNNSLFLDYHRNPFPVFFLRGLHVSC STDDPLQIHLTKEPLVEEYSIAASVWKLSACDLCEIIRESVHHSGFSHALFSHWIGKDYYQRGPDGNDIH KTNVPHIRVEFRDTIWKEEMQQVYLGKAVISDEVEH 31 MLPNTGRLAGCTVFITGASRGIGKAIALKAAKDGANIVIAAKTAQPHPKLLGTIYTAAEEIEAVGGKALP CIVDVRDEQQISAAVEKAIKKFGGIDILVNNASAISLTNTLDTPTKRLDLMMNVNTRGTYLASKACIPYL KKSKVAHILNISPPLNLNPVWFKQHCAYTIAKYGMSMYVLGMAEEFKGEIAVNALWPKTAIHTAAMDMLG GIESQCRKVDIIADAAYSIFQKPKSFTGNFVIDENILKEEGIENFDVYAIKPGHPLQPDFFLD 32 MLPNTGRLAGCTVFITGASRGDGKAIALKAAKDGANIVIAAKTAQPHPKLLGTIYTAAEEIEAVGGKALP CIVDVRDEQQISAAVEKAIKKFGGIDILVNHAEAISLTNTLDTPTKRLDLMMNVNTRGTYLASKACIPYL KKSKVAHILNHSPPLNLNPVWFKQHCAYTIARYGMSMYVLGMAEEFKGEIAVNALWPKTAIHTAAMDMLG GIESQCRKVDIIADAAYSIFQKPKSFTGNFVIDENILKEEGIENFDVYAIKPGHPLQPDFFLD 33 MLPNTGRLAGCTVFITGASRGDGKNIAQKAAKDGANIVILAKTAQPHPKLLGTIYTAAEEIEAVGGKALP CIVDVRDEQQISAAVEKAIKKFGGIDILVNNAEAINLTNTLDTPTKRLDLMMWVNTRGPYLASKACIPYL KKSKVAHILNHSPPLNLNPVWFKQHCAYTIARYGMSMYVLGMAEEFKGEIAVNAHWPYTAIHTAAMDMCG GIESQCGKVDIIAWAAESIFQKPKSFTGNFVINENILKEEGIENFDVYAIKPGHPLQPDFFLD 34 MLSPAVQTFWKWLQEEGVITAKTPVKASVVTEGLGLVALKDISRNDVILQVPKRLWINPDAVAASEIGRV CSELKPWLSVILFLIRERSREDSVWKHYFGILPQETDSTIYWSEEELQELQGSQLLKTTVSVKEYVKNEC LKLEQEIILPNKRLFPDPVTLDDFFWAFGILRSRAFSRLRNENLVVVPMADLINHSAGVTTEDHAYEVKG AAGLFSWDYLFSLKSPLSVKAGEQVYIQYDLNKSNAELALDYGFI 35 MLSPAVQTFWKWLQEEGVITAKTPVKASVVTEGLGLVALKDISRNDVILQVPKRLWINPDAVAASEIGRV CSELKPWLSVILFLIRERSREDSVWKHYFGILPQETDSTIYWSEEELQELQGSQLLKTTVSVKEYVKNEC LKLEQEIILPNKRLFPDPVTLDDFFWAFGILRHRAESRLRNENLVVVPMADLEEHSAGVTTEDHAYEVKG AAGLFSWDYLFSLKSPLSVKAGEQVYIQGDLNKSNAELALDHGFI 36 MLSPAVQTFWKWLQEEGVITAKTPVKASVVTEGLGHVALKDISRNDVILQVPKRLWINPDAVAASEIGRV CSELKPWLSVILFLIREQSKEDSRWKHYFGILPWETDKTIYWSEEELQELQGSQLLKTTVSVKEYVKNEC LKLEQEIILPNKRLFPDPVTLDDFFWAFGMCRHRAESRLRNENLVVVPMADLEEHSAGQTAEHHAYEVCG AAGQNSWDYLFSWTEPLNVKAGFQVYIQGDLIKSNAELYLDRGFI 37 MPFPYEFRELNPEEDKLVKANLGAFPTTYVKLGPKGYMVYRPYLKDAANIYNMPLRPTDVFVASYQRSGT TMTQELVWLIENDLNFEAAKTYMSLRYIYLDGFMIYDPEKQEEYNDILPNPENLDMERYLGLEYSSRPGS SLLAAVPPTEKRFVKTHLPLSLMPPNMLDTVKMVYLARDPRDVAVSSFHHARLLYLLNKQSNFKDFWEMF HRGLYTLTPYFEHVKEAWAKRHDPNMLFLFYEDYLKDLPGCIARIADFLGKKLSEEQIQRLCEHLNFEKF KNNGAVNMEDYREIGILADGEHFIRKGKAGCWRDYFDEEMTKQAEKWIKDNLKDTDLRYPNM 38 MPFPYEFRELNPEEDKLVKANLGAEPTTYVKLGPKGYMVYRPYLKDAANIYNMPLRPTDVFVASYQRSGT TMTQELVWLIENDLNFEAAKTYMSLRYIYLDGFMRYDPEKQEEYNDELPNPENLDMERYLGLEYSSRPGS SLLAAVPPTEKRFVKTHLPLSLMPPNMLDTVKMVYLARDPRDVAVSSFHHARLDYELNKQSNFKDFWEMF HRGLYTLTPYFEHVKEAWAKRHDPNMLFLFYEDYLKDLPGCIARIADFLGKKLSEEQIQRLCEHLNFEKF KNNGAVNMEDEREIGHEADGEHFIRKGKAGCWRDYFDEEMTKQAEKWIKDNLKDTDLRYPNM 39 MPFPYEFRELNPEEDKLVKANLDSEPDTEVKLGPKGYMVYFPDLKDAANIYNMPLRPTDVFVASYQRTGT TMTQELVWLIENDLNFEAAKTYMSLRYIYDDGFMRYFPEKLEAYNFELMIPRKPMSEKRYFLEGTVCIGS SLLAAVPPTEKRFVKTDLELSLMPPNMLDTVKMVYLARDPRDVAVSSFGNYRRDYSLCIQSNFKDFWEMF HRGLYTLVPYFEHVKEAWAKRHDPNMLFLFYEDYLKDLPGCIARIADFLGKKLSEEQIQRLCEHLNFEKF KNNGAVNMHDTRGQGVEADMIHRISKGKAGCWRDYFDEEMTKQAEKWIKDNLKDTDLRYPNM 40 MKIPKDTLIIAVENEIARINPAYSEDHDAVINLVFSGLTRFDENMSLKPDLAKSWDISKDGLVYDIFLRD DVLWHDGVKFSADDVKFSIEAFKNPKNNSSIYVNFEDIKSVEILNPSHVKITLFKPYPAFLDALSIGMLP KHLLENENLNTSSFNQNPIGTGPYKFVKWKKGEYVEFKANEHFYLDKVKTPRLIIKHIFDPSIASAELKN GKIDAALIDVSLLNIFKNDENFGILREKSADYRALMFNLDNEFLKDLKVRQALNYAVDKESIVKNLLHDY AFVANHPLERSWANSKNFKIYKYDPKKAEDLLVSAGFKKNKDGNFEKDGKILEFEIWAMSNDPLRVSLAG ILQSEFRKIGVVSKVVAKPAGSFDYSKVDSFLIGWGSPLDPDFHTFRVFESSQDSALNDEGWNFGHYHDK KVDIALQKARNTSNLEERKKYYKDFIDALYENPPFIFLAYLDFALVYNKDLKGIKTRTLGHHGVGFTWNV YEWSK 41MKIPKDTLIIAVENEIARINPAYSEDHDAVINLVF SGLTRFDENMSLKPDLAKSWDISKDGLVYDIFLRDDVLWHDGVKFSADDVKFSIEAFKNPKNNSSIYVNF EDIKSVEILNPSHVKITLFKPYPAFLDALSIGMLPKHLLENENLNTSSFNQNPIGTGPYKFVKWKKGEYV EFKANEHFYLDKVKTPRLIIKHIFDPSIASAELKNGKIDAALIDVSLLNIFKNDENFGILREKSADYRAL MFNLDNEFLKDLKVRQALNYAVDKESIVKNLLHDYAFVANHPLERSWANSKNFKIYKYDPKKAEDLLVSA GFKKNKDGNFEKDGKILEFEIWAMSNDPLRVSLAGILQSEFRKIGVVSKVVAKPAGSFDYSKVDSFLEGH GSPLDPDFHTFRVFESSQDSALNDEGENRGHYHDKKVDIALQKARNTSNLEERKKYYKDFIDALYENPPF IFLAYLDFALVYNKDLKGIKTRTLGHHGVGFTWNVYEWSK 42 MKIPKDTLIIAVVNEPSRINPAYSWDHDCKANLVFSGLTRFDENMSLKPDLAKSWDISKDGLVYDIFLRD DVLWHDGVKFSADDVKFSIEAFKNPKNNSTQCKWAEDIKSVEILNPSHVKITLFKPYPAFLDALSIGMLP KHLLENENLNTSSFNQNPIGTGPYKFVKWKKGEYVEFKANEHFYLDKVKTPRLIICEMNQMFIASAELKN GKIDAALIDVSLLNIFKNDENFGILREKSADYRALMFNLDNEFLKDLKVRQALNYAVDKESIVKNLLHDY AFVANHPLERSWANSKNFKIYKYDPKKAEDLLVSAGFKKNKDGNFEKDGKILEFEIWQHSADPLRVSLAG ILQSEFRKIGVVSKVVAKPRCVFDGKCVDSFLEGHGSPLDPDFHTFRVFESSQDSALNDDDANYGSDTDK KVDIALQKARNTSNLEERKKYYKDFIDALYENPPFIFLAYLDFALVYNKDLKGIKTRTLGHHGVGFTWNV YEWSK 43MALKFNPLVSQPYKLASSARPPVSTFRSPKFLCLA SSSSPALSSKEVESLKKPFTPPREVHLQVLHSMPPQKIEIFKSMEDWAEQNLLPHLKDVEKSWQPQDFLP DPASDGFEDQVKELRERARELPDDYFVVLVGDMITEEALPTYQTMLNTLDGVRDETGASPTSWAVWTRAW TAEENRHGDLLNKYLYLSGRVDMRQIEKTIQYLIGSGMDPRTENNPYLGFIYTSFQERATFVSHGNTARQ AKEHGDLKLAQICGTIAADEKRHETAYTKIVEKLLEIDPDGTVVAFADMMRKKISMPAHLMYDGRDDNLF DNFSSVAQRLGVYTAKDYADILEFLVGRWRIESLTGLSGEGNKAQEYLCGLTPRIRRLDERAQARAKKGP KIPFSWIHDREVQLVD 44MPAHLLQEEISSSYTTTTTITAPPSRVLQNGGGKL EKTPLYLEEDIRPEMRDDIYDPTYQDKEGPKPKLEYVWRNIILMSLLHLGALYGITLIPTCKIYTYIWVL FYYLMGALGITAGAHRLWSHRTYKARLPLRVFLIIGNTMAFQNDVFEWSRDHRAHHKFSETDADPHNSRR GFFFSHVGWLLVRKHPAVKEKGSTLNLSDLRAEKLVMFQRRYYKPGVLLLCFILPTLVPWYLWDETFQNS LFFATLFRYALGLNVTWLVNSAAHMYGYRPYDKTINPRENILVSLGAAGEGFHNYHHTFPYDYSASEYRW HINFTTFFIDCMAAIGLAYDRKKVSKAAILARIKRTGEESYKSGVD 45 MALKLNPFLSQTQKLPSFALPPMASTRSPKFYMASTLKSGSKEVENLKKPFMPPREVHVQVTHSMPPQKI EIFKSLDNWAEENILVHLKPVEKCWQPQDFLPDPASDGFDEQVRELRERAKEIPDDYFVVLVGDMITEEA LPTYQTMLNTLDGVRDETGASPTSWAIWTRAWTAEENRHGDLLNKYLYLSGRVDMRQIEKTIQYLIGSGM DPRTENSPYLGFIYTSFQERATFISHGNTARQAKEHGDIKLAQICGTIAADEKRHETAYTKIVEKLFEID PDGTVLAFADMMRKKISMPAHLMYDGRDDNLFDHFSAVAQRLGVYTAKDYADILEFLVGRWKVDKLTGLS AEGQKAQDYVCRLPPRIRRLEERAQGRAKEAPTMPFSWIFDRQVKLVD 46 MALKLCFPPHKMPSFPDARIRSHRVFMASTIHSPSMEVGKVKKPFTPPREVHVQVTHSLAPEKREIFNSL NNWAQENILVLLKDVDKCWQPSDFLPDSASEGFDEQVMELRKRCKEIPDDYFIVLVGDMITEEALPTYQT MLNTLDGVRDETGASLTPWAIWTRAWTAEENRHGDLLNKYLYLSGRVDMKQIEKTIQYLIGSGMDPRTEN NPYLGFIYTSFQERATFISHGNTARLAKEHGDLKLAQICGIIAADEKRHETAYTKIVEKLFEIDPDGTVL ALADMMRKKVSMPAHLMYDGQDDNLFENFSSVAQRLGVYTAKDYADILEFLVGRWDIEKLTGLSGEGRKA QDYVCTLPPRIRRLEERAQSRVKKASATPFSWIFGREINLVD 47 MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRALHPGNIPNRDWAVEYYTQRAQRPGTMII TEGAFISPQAGGYDNAPGVWSEEQMVEWTKIFNAIHEKKSFVWVQLWVLGWAAFPDNLARDGLRYDSASD NVFMDAEQEAKAKKANNPQHSLTKDEIKQYIKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNTR TDEYGGSIENRARFTLEVVDALVEAIGHEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVAGELEKRAKAG KRLAFVHLVEPRVTNPFLTEGEGEYEGGSNDFVYSIWKGPVIRAGNFALHPEVVREEVKDKRTLIGYGRF FISNPDLVDRLEKGLPLNKYDRDTFYQMSAHGYIDYPTYEEALKLGWDKK 48 MASMVAFRPEAFLCFSPPKTTRSTRSPRISMASTVGPSTKVEIPKKPFMPPREVHVQVTHSMPPQKIEIF KSLEDWAENNILVHLKPVEKCWQPQDFLPDPSSEGFHEEVKELRERSKEIPDDYYVCLVGDMITEEALPT YQTMLNTLDGVRDETGASLTSWAVWTRAWTAEENRHGDLLNKYLYLSGRVDMKQIEKTIQYLIGSGMDPR TENSPYLGFIYTSFQERATFISHGNTARHAKEHGDVKLAQICGTIASDEKRHETAYTKIVEKLFEIDPDG TVLSFADMMKKKISMPAHLMYDGQDDNLFEHFSAVAQRLGVYTAKDYADILEFLINRWKVGELTGFSGEG KRAQDFVCTLAPRIRRIEERAQERAKQAPRIPFSWIYGREVQLVD 49 MEQPQAMEKVESVDVTEANAVAAGTNKLTKRIVAAGLGGKLMGTKSMVDVTADQLAKDSINELLARDKAL KEKYEKQKHISEQPWTKDNWHQHINWLNMILVCGLPLFGAIATYYKPPTKETVILGVVLYILGGLSITAG YHRLWSHRAYSARTPLKVLFALFGAGAVEGSIKWWSHSHRVHHRYTDTHRDPYDARKGFWYSHMGWMLTK PNPRYKARADISDLADDWIVRFQHRHYLLIMTFMALILPTIIAKYFWDDAWGGFIYGGIFKVFVIQQATF CVNSLAHWIGVQPFDDRRTPRDHFLTAIVTFGEGYHNFHHEFPSDYRNALKWYQYDPTKIVIYLSSKVGL SYNLKTFSDNAIKQGLVQQQQKKLDSMRAHLNWGTPLQDLPVWDKSEFFEKSKDKKGLVIISGIVHNVEN FIGEHPGGEKLVKGALGKDATTAFNGGVYAHSNAAHNLLATMRVAVIRDGNANANTFSLQNEFLDKKAAA AAASN 50MDIPTAKYSEEDMKAALQPTEPEFFKVKVSKKKKN RRKVKHDTTASTIRRHISESKWTISNWYRHIHWKNVVLVVIVPLLALLGSLTVPLTRTGFYWMGFNYIIT CTFVIMGYHRYWAHRSFQTTKEMSFLFAMVGASAGVGSAKWWCASHRAHHRFCDTERDPHNIRKGFWYSH FGWMLLIHHPKVQAAIKESESEDISSDPVILWQYENYFQMFLVTGILIPAIIGWWLFQDFAGGLVYGGLV KIFLVQNTIFSVNSLCHCCGSQPFDDAKSSRNSILLSLITFGEGQHNFHHEFPSDCRNGVEWYDFDPVKW VIFTLNALGVIYNVHAAPTTAINQLRVQQAQRILDKERSQLNWGIRIDKLPTLTSAEFSKLAKESQPQRA LVVISGIVHDVTPFLHDHPGGVALIKSSIGKDATNAFNGAVYSHSNAARNLLATMRIAVILGSEQEVWKQ QQKENKDVPLKNDSEGKKIVRSGEQITMTKTPSTTAGAA 51 MSAVTVTGNNGDASRSNTTTTTKRTGNVSSFSQSKGLTAIDTWGNVFKVPDFTIKQILDAIPKHCYERRL TTSFYYVFRDIFLIGCTMFMGSFIPMIENVFLRGAAYAALVFLLSVEYTGLWVLAHECGHQAFSDYGWVN DTVGWILHSYLLVPYFSWKYSHGKHHKATGHLTRDMVFVPATKEKFLEKRNASKLGELGEDAPIFTLYQL VAQQLGGWILYLFTNVTGQPYPNTPKWMQNHFVPSSPIFEKKDYWFIILSDLGILAQLMVLYVWRQQMGN WNLFIYWFLPYVLTNHWLVFITFLQHSDPTMPHYEAEQWTFARGAAATIDREFGFIGPFFFHDIIETHVL HHYVSRIPFYNAREASEGIKKVMGEHYRYSGENMWVSLWKSGRSCQFVDGENGVKMYRNINNWGIGTGEK 52MSKVTVSGSEILEGSTKTVRRSGNVASFKQQKTAI DTFGNVFKVPDYTIKDILDAIPKHCYERSLVKSMSYVVRDIVAISAIAYVGLTYIPLLPNEFLRFAAWSA YVFSISCFGFGIWILGHECGHSAFSNYGWVNDTVGWVLHSLVMVPYFSWKFSHAKHHKATGHMTRDMVFV PYTAEEFKEKHQVTSLHDIAEETPIYSVFALLFQQLGGLSLYLATNATGQPYPGVSKFFRSHYWPSSPVF DKKDYWYIVLSDLGILATLTSVYTAYKVFGFWPTFITWFCPWILVNHWLVFVTFLQHTDSSMPHYDAQEW TFAKGAAATIDREFGILGIIFHDIIETHVLHHYVSRIPFYHAREATECIKKVMGEHYRHTDENMWVSLWK TWRSCQFVENHDGVYMFRNCNNVGVKPKDT

In some embodiments, the enzyme catalyst includes an enzyme having anamino acid sequence as set forth in any one of SEQ ID NOs: 1-52, or anyamino acid sequence having a sequence identity to any one of SEQ ID NOs:1-52 of at least 75%, such as 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence homology to any one of SEQ ID NOs:1-52, or a sequence homology within a range defined by any two of theaforementioned values.

In some embodiments, the enzyme catalysts are used in a solublesuspension or are immobilized to a solid substrate. In some embodimentsthe enzyme catalysts are immobilized to suitable polymeric supports byadsorption, affinity interactions, or covalent bonding. In someembodiments, the enzyme catalysts are self-immobilized through theformation of cross-linked aggregates, or entrapped into insolublepolymer microbeads. All of these immobilization approaches can beperformed either by the natural physicochemical properties of the enzymecatalysts, or by addition of specific protein tags that allow theseimmobilization processes.

In some embodiments, the enzyme catalyst is an engineered desaturaseenzyme. In some embodiments, the enzyme catalyst is a modified proteinof the fatty acid desaturase group of enzymes that can be engineered tointeract with a wider range of lipid molecules including mono-, di-, ortriglycerides (as shown in FIG. 2 ). In some embodiments, the enzymecatalyst performs hydrogenase activity by metal substitution fortransition metals, such as nickel, iron, palladium, or other transitionmetal ions.

In some embodiments, metal substitution can be performed by variousapproaches, including for example: removal of the native metal by ametal binding agent and/or competition with hydrogen ions at low pH,followed by insertion of the new metal; direct competition of the firstmetal by a different metal; and/or biosynthesis of the protein underenriched conditions of the metal of choice, such as nickel, iron,palladium, or other transition metal.

In some embodiments, oxidation of molecular hydrogen (H₂) and metalsubstitution can occur based on the mechanism shown in FIG. 3 . Withoutwishing to be bound by theory, the mechanism depicted may be based onthe molecular hydrogen oxidation by hydrogenases, in which dissociatedprotons have the potential to form hydride ions with the ion metalslocated inside the protein core, later promoting the oxidation andconsequent reduction of a double carbon bond. The metal centers of theproteins are stabilized by residues such as histidines and cysteines,and the affinity of the protein toward metal ions can be improved bypoint and sequential amino acid substitutions of proximal residues tothe metal core, by residues with high affinity toward transition metals,such as cysteine, histidine, aspartic acid, and/or glutamic acidresidues.

In some embodiments, the methods provided herein may further include theaddition of a base, including a strong base. In some embodiments,addition of a base, such as arginine (R), to the active site can help inthe reaction. Without wishing to be bound by theory, embodiments thatinclude addition of a base may result in formation of a Frustrated Lewispair reaction, which can be developed on site (FIG. 4 ), where the twometal ions (that act as a Lewis acid) may work in conjunction with thearginine (that works as the Lewis base) to split H₂ in 2H+.

The concept of Lewis acidity and basicity and the formation of simpleLewis acid-base adducts is a primary axiom of main group chemistry.(Lewis, Valence and the Structure of Atoms and Molecules, ChemicalCatalogue Company, Inc., New York, 1923). The combination of Lewisdonors and acceptors in which steric demands preclude formation ofsimple acid-base adducts have been termed “frustrated Lewis pairs”(FLPs) (Stephan, Org. Biomol. Chem., 2008, 6, 1535-1539).

Results from the biochemical saturation process with the engineereddesaturase enzymes are shown in FIGS. 8A and 8B, and FIG. 9 . As usedherein, the term “canonical” has its ordinary meaning as understood inlight of the specification, and refers to a type that is typically foundin a class. For example, a canonical substrate is a substrate that istypically found among substrate. Conversely, a non-canonical substrateis one that differs from canonical substrate, such as one that is nottypical of the type of substrates.

In some embodiments, the enzyme catalyst is an engineered hydrogenaseenzyme. In some embodiments, the enzyme catalyst may be a modifiedhydrogenase to perform hydration of lipids by engineering a lipidbinding region next to the metal core region (FIG. 5 ).

In some embodiments, the enzyme catalyst is a novel enzyme. In someembodiments, the enzyme catalyst may be a novel designed enzyme,containing a hydrogenase active site and a lipid-binding site.

In some embodiments, the hydrogenase active site includes a [Ni—Fe],[Fe—Fe], or [Ni—Ni] metal core, and an opposite arginine. In someembodiments, the metal binding site includes transition metals, such asnickel, iron, palladium, cobalt, scandium, vanadium, chromium,manganese, molybdenum, rhodium, or other transition metal.

In some embodiments, the metal atoms bonded to the active site cancapture molecular hydrogen gas molecules, orienting one of the hydrogenatoms towards the positively charged amino group of the arginineresidue, producing a frustrated Lewis pair reaction splitting thehydrogen molecule into two hydrogen atoms. These hydrogen atoms are thentransferred to the carbon-carbon double bond of the near lipid bonded tothe lipid-binding active site, releasing a saturated lipid (FIG. 6 ).

The lipid binding pocket can be designed in order to position the targetcarbon bond next to the hydrogenase site (FIG. 7A), and in this waydifferent novel enzymes can be designed in order to saturate specificcarbon atoms in the carbon chain.

In some embodiments, the enzyme catalyst is an engineered reductaseenzyme. In some embodiments, the enzyme catalyst may be a lipidreductase enzyme, which normally reduces free fatty acids using redoxcofactors (NADH, NADPH, FADH₂), that can be engineered to interact witha wider range of lipid molecules including mono-, di- and triglycerides.FIG. 7A depicts a representation of a protein, such as any of theproteins described in Table 1. The protein includes an active site,which includes a catalytic core, and an amino acid opposite thecatalytic core. In some embodiments, the catalytic core is a metal core.In some embodiments, the catalytic core includes a nickel and an ironatom (Ni—Fe core). In some embodiments, the opposite amino acid is anarginine. In some embodiments, the active site is configured to bind asubstrate, such as a lipid. In some embodiments, the lipid is oleicacid. As shown in FIG. 7B, nickel binding in a designed pocket greatlyreduces the intrinsic protein fluorescence in the novel enzyme design(protein 3-SEQ ID NO: 3), but not to the same degree in a design lackingthe nickel binding domain (protein 1-SEQ ID NO: 1).

In some embodiments, the methods include a post-hydrogenation process.In some embodiments, the product obtained from any of the hydrogenationreactions described herein are processed through a variety of methods inorder to remove unwanted elements remnant from the enzymatic reaction,such as salts, leached metal particles, water, and water solublecomponents. These processing methods may include, for example, organicsolvent extraction, sedimentation, filtration, vacuum treatment,centrifugation, and/or particle adsorption. These processes allow torecover all the fatty compounds, leaving the oil free of aqueous-phaseor water-soluble components. In some embodiments, the methods allowrecovery of target compounds. In some embodiments, unwanted organicsolvents can also be removed from the processed product by rotavapor,distillation, vacuum treatment, or a combination thereof.

Additionally, the product obtained from the enzymatic saturation of oilscan be further enriched in saturated lipids by dry fractionation,solvent-based fractionation, detergent-assisted fractionation,distillation, supercritical extraction or a combination of thesemethods. The product of enzymatic saturation of oils can be used as wellin interesterification and oil-blending processes to further fine tunethe physical properties of the product.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure. Those in the art willappreciate that many other embodiments also fall within the scope of thedisclosure, as it is described herein above and in the claims.

Example 1: Production of Partially Hydrogenated Fatty Acid

The following example demonstrates an example method for producing apartially hydrogenated fatty acid.

An unsaturated starting material was obtained. Hydrogen gas wasintroduced into a reaction chamber, together with an enzyme catalyst(one or more of a hydrogenase enzyme, including, for example, any enzymehaving an amino acid sequence as set forth in SEQ ID NOs: 1-52, or anenzyme having a sequence that is at least 75% identical to any one ofSEQ ID NOs: 1-52, such as 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-52).The starting material was added to the reaction chamber together withthe hydrogen gas and the enzyme catalyst for a period of time sufficientto catalyze the starting material to at least partially hydrogenatedproducts (partially saturated product).

In some embodiments, the enzyme catalyst was an engineered enzymecatalyst such as an enzyme catalyst as provided herein. In someembodiments, the enzyme catalyst included a metal core, such as anickel-iron [Ni—Fe] core, and an opposite arginine. In some embodiments,the enzyme catalyst has an amino acid sequence as set forth in SEQ IDNO: 3.

The starting material was any suitable starting material, including, forexample, any partially of fully unsaturated molecule, such asmyristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidicacid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenicacid, arachidonic acid, erucic acid, docosahexaenoic acid, andeicosapentaenoic acid. The saturated product may be any equivalentsaturated product, including, for example, butyric acid, lauric acid,myristic acid, palmitic acid, or stearic acid.

Example 2: Use of Desaturase Enzymes to Saturate Vegetable Oils

The coding sequence for the Olea europaea oleate desaturase enzyme(OLΔ9) was optimized for recombinant production in Pichia pastoris, andthe obtained synthetic DNA was cloned downstream a methanol-induciblepromoter for the recombinant production of the enzyme by secretion tothe extracellular media. The production of OLΔ9 was monitored byconcentrating the clarified culture supernatants by ammonium sulfateprecipitation, followed by SDS-PAGE electrophoresis.

In order to detect the canonical desaturase enzymatic activity in theobtained precipitates, these were resolubilized by dialysis againstphosphate buffer saline solution, and then mixed together with 0.05%Tween 20, 5 mM DTT and 1% stearic acid, and incubated at 30° C. for 24h. After this time, fatty acids were extracted with ethyl acetate, andthe saturation status assessed by iodine value. As shown in FIG. 8A,increasing concentrations of enzyme preparations generated thedesaturation of stearic acid, reflected in the increase in iodine value.

The non-canonical saturation with the OLΔ9 enzyme was tested byrecreating the conditions described above, but in a vessel saturatedwith hydrogen gas. Stearic acid was replaced with 90% olive oil, and thereaction was performed for a time period of 5 hours. As shown in FIG.8B, iodine value of the extracted oil showed a decrease in saturationlevel. Additionally, two phases of the oil were obtained, with an upperfraction showing a solid, higher melting point behavior (FIG. 9 )

Example 3: Use of Engineered Hydrogenase Enzymes to Saturate Free FattyAcids and Vegetable Oils

The coding sequence for engineered hydrogenase enzymes Protein-ID #6(SEQ ID NO: 6), Protein-ID #7 (SEQ ID NO: 7), and Protein-ID #8 (SEQ IDNO: 8) were optimized for recombinant production in Escherichia coli,fused to 6×HIS sequence for immobilized metal affinity chromatography(IMAC), and the obtained synthetic DNA was cloned downstream anIPTG-inducible promoter for the recombinant production of the enzyme.Enzymes of 35 kDa were readily detected in the insoluble (I) fractionsof cell lysates (FIG. 10A), confirming recombinant expression.

Protein-ID #8 (SEQ ID NO: 8) was chosen for saturation of oleic acid. Tothis end, after induction of recombinant Protein-ID #8 (SEQ ID NO: 8)expression, E. coli cells were lysed by sonication, and the insolubleprotein fraction recovered by centrifugation. Proteins were solubilizedin a buffer that included 8 M urea, 0.1% Triton X-100, 5 mM DTT, 100 mMTris buffer and Protein-ID #8 (SEQ ID NO: 8) purified by IMAC. Afterelution, the obtained proteins were subjected to dialysis in refoldingbuffer (20 mM HEPES, 300 mM NaCl, 0.5 M trehalose, at a pH of 7.5), witha stepwise decrease in urea concentration to allow renaturation ofProtein-ID #8 (SEQ ID NO: 8). Finally, the protein was lyophilized andlater used in hydrogenation reactions. Briefly, proteins were mixed with0.01 g of NiCl₂·6 H₂O, 0.1 g of oleic acid, 30 mL of chloroform, and 10mL of water in a boiling flask, and incubated at 40° C. for a timeperiod of 8 hours, in an atmosphere saturated with H₂ gas. After thisperiod, the reaction mixture was cooled to room temperature and thevolatiles were evaporated under vacuum. As a control, the same reactionwas run with all components except Protein-ID #8 (SEQ ID NO: 8). Thecrude products were filtered through celite using ethyl acetate and thevolatiles were evaporated under vacuum. The obtained samples wereanalyzed by GC-FID analysis, and fatty acid composition compared to anunsaturated oleic acid sample (FIG. 10B).

The obtained results surprisingly showed that when NiCl₂ was used alonefor the saturation reaction, no significant changes were observedcompared to the unsaturated oleic acid control. In contrast, whenProtein-ID #8 (SEQ ID NO: 8)+NiCl₂ was used as a catalyst, a significantdecrease in oleic acid, and concomitant increase in stearic acid wasobserved. No changes in trans fatty acids were observed.

Example 4: Use of Metal Binding Enzymes to Saturate Vegetable Oils

The coding sequence for Protein-ID #40 (SEQ ID NO: 40) was optimized forrecombinant production in Escherichia coli, and the obtained syntheticDNA was cloned downstream an IPTG-inducible promoter for the recombinantproduction of the enzyme. An enzyme of 53 kDa was readily detected inthe soluble (S) and insoluble (I) fractions of cell lysates (FIG. 11A),confirming good recombinant expression.

A second generation of engineered enzymes was also developed in order togenerate variants adding one additional nickel binding site (Protein-ID#41-SEQ ID NO: 41) and also improving the affinity towards free fattyacids (Protein-ID #42-SEQ ID NO: 42). When these were expressed in arecombinant manner as described above, good protein expression was alsodemonstrated (FIG. 11B).

The ability of Protein-ID #40 (SEQ ID NO: 40) to be used to saturatecanola oil was tested. To this end, after induction of expression ofrecombinant Protein-ID #40 (SEQ ID NO: 40) in E. coli, cells were lysedby sonication, centrifuged to remove insoluble material, and metalbinding proteins of interest purified by immobilized metal affinitychromatography. After elution, the obtained proteins were subjected todialysis against HEPES buffer to remove unwanted salts and metals, andlyophilized. The obtained proteins were then used in hydrogenationreactions. Briefly, proteins were mixed with 0.01 g of NiCl₂·6 H₂O, 0.1g of canola oil, 30 mL of chloroform, and 10 mL of water in a boilingflask, and incubated at 40° C. for 8 hours, in an atmosphere saturatedwith H₂ gas. After this period, the reaction mixture was cooled to roomtemperature and the volatiles were evaporated under vacuum. The crudeproduct was filtered through celite using ethyl acetate and thevolatiles were evaporated under vacuum. The obtained sample was analyzedby GC-FID analysis, and fatty acid composition compared to anunsaturated canola oil sample (FIG. 11C).

The obtained results unexpectedly showed that the reaction allows thepartial hydrogenation of canola oil, significantly decreasing theconcentration of linoleic acid (C18:2), increasing oleic acidconcentration (C18:1), and avoiding any increase in trans fatty acids.These results show that this method can generate stereospecificcatalytic hydrogenation of vegetable oils, and avoid the generation oftroublesome trans fatty acids.

Example 5: Use of Novel Enzyme to Partially Saturate Canola Oil

The protein sequence for natural Protein-ID #13 (SEQ ID NO: 13) wasmodified to contain a metal binding domain, and further modified inorder to increase its affinity towards free fatty acids, generatingnovel enzyme Protein-ID #15 (SEQ ID NO: 15). The coding sequences forthese proteins were optimized for expression in Escherichia coli, andthe obtained synthetic DNA cloned downstream an IPTG-inducible promoter,and upstream of a in frame 6×HIS tag for the recombinant production ofthe enzyme. Protein-ID #13 (SEQ ID NO: 13) and #15 (SEQ ID NO: 15) of 31and 32 kDa respectively could be readily detected in the insoluble (I)fractions of cell lysates (FIG. 13A), confirming good recombinantexpression, although Protein-ID #13 (SEQ ID NO: 13) showed a sizeslightly smaller than expected.

The ability of Protein-ID #15 (SEQ ID NO: 15) to be used to partiallysaturate canola oil was put to a test. To this end, after induction ofexpression of recombinant Protein-ID #15 in E. coli, cells were lysed bysonication, and the insoluble protein fraction recovered bycentrifugation. Proteins were solubilized in an 8 M urea, 0.1% TritonX-100, 5 mM DTT, 100 mM Tris buffer and Protein-ID #15 (SEQ ID NO: 15)purified by IMAC. After elution, the obtained proteins were subjected todialysis in refolding buffer (20 mM HEPES, 300 mM NaCl, 0.5 M Trehalose,pH 7.5), with a stepwise decrease in Urea concentration to allow proteinrenaturation. Finally, the protein was lyophilized and later used inhydrogenation reactions: 68 mg of pure Protein-ID #15 (SEQ ID NO: 15)was mixed with 0.01 g of NiCl₂·6 H₂O, 0.1 g of canola oil, 15 mL ofchloroform and 6 mL of water in a boiling flask, and incubated at 40° C.for 8 hours, in an atmosphere saturated with H₂ gas. After this period,the reaction mixture was cooled to room temperature and the volatileswere evaporated under vacuum. The crude products were filtered throughcelite using ethyl acetate and the volatiles were evaporated undervacuum. The obtained samples were analyzed by GC-FID analysis, and fattyacid composition compared to an unsaturated oleic acid sample (FIG.13B).

The obtained results show that the reaction allows the partialhydrogenation of canola oil, decreasing the concentration of linoleicacid (C18:2), increasing oleic acid concentration (C18:1) and avoidingany increase in trans fatty acids. These results show that this Proteinand method can generate stereospecific catalytic hydrogenation ofvegetable oils, and avoid the generation of troublesome trans fattyacids.

With respect to the use of plural and/or singular terms herein, thosehaving skill in the art can translate from the plural to the singularand/or from the singular to the plural as is appropriate to the contextand/or application. The various singular/plural permutations may beexpressly set forth herein for sake of clarity.

It will be understood by those of skill within the art that, in general,terms used herein, and especially in the appended claims (e.g., bodiesof the appended claims) are generally intended as “open” terms (e.g.,the term “including” should be interpreted as “including but not limitedto,” the term “having” should be interpreted as “having at least,” theterm “includes” should be interpreted as “includes but is not limitedto,” etc.). It will be further understood by those within the art thatif a specific number of an introduced claim recitation is intended, suchan intent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

Any of the features of an embodiment of the first through second aspectsis applicable to all aspects and embodiments identified herein.Moreover, any of the features of an embodiment of the first throughthird aspects is independently combinable, partly or wholly with otherembodiments described herein in any way, e.g., one, two, or three ormore embodiments may be combinable in whole or in part. Further, any ofthe features of an embodiment of the first through third aspects may bemade optional to other aspects or embodiments.

What is claimed is:
 1. A method of saturating an unsaturated molecule, the method comprising: contacting an unsaturated molecule with an enzyme to produce a saturated molecule; and recovering the saturated molecule.
 2. The method of claim 1, wherein the saturated molecule is fully or partially saturated.
 3. The method of claim 1, wherein the unsaturated molecule comprises an unsaturated alkene.
 4. The method of claim 1, wherein the unsaturated molecule is an unsaturated triglyceride or a free fatty acid.
 5. The method of claim 1, wherein the unsaturated molecule is vegetable oil.
 6. The method of claim 1, wherein the unsaturated molecule is olive oil or canola oil.
 7. The method of claim 1, wherein contacting the unsaturated molecule with the enzyme is performed in a solvent.
 8. The method of claim 1, wherein the contacting is performed for a sufficient period of time to allow at least partial saturation.
 9. The method of claim 1, wherein the enzyme is in solution, or wherein the enzyme is immobilized.
 10. The method of claim 9, wherein the enzyme is immobilized on a polymeric support.
 11. The method of claim 10, wherein the polymeric support is an insoluble polymer microbead.
 12. The method of claim 1, wherein the enzyme is prepared by protein fermentation or chemical synthesis.
 13. The method of claim 1, wherein the enzyme is a purified enzyme.
 14. The method of claim 1, wherein the enzyme is a recombinant nickel binding enzyme.
 15. The method of claim 1, wherein the enzyme comprises a consensus sequence as set forth in SEQ ID NO:
 53. 16. The method of claim 1, wherein the enzyme has an amino acid sequence as set forth in SEQ ID NOs: 1-52, or having a sequence identity of at least 75% to any one of SEQ ID NOs: 1-52.
 17. The method of claim 1, wherein the enzyme has an amino acid sequence as set forth in SEQ ID NO: 15 or 40, or having a sequence identity of at least 75% to any one of SEQ ID NOs: 15 or
 40. 18. The method of claim 1, wherein the enzyme is a novel designed protein having hydrogenase activity and comprising a substrate specific binding site.
 19. The method of claim 18, wherein said substrate specific binding site comprises one or more alkene unsaturation sites.
 20. The method of claim 1, wherein said enzyme is a hydrogenase enzyme engineered to bind a non-canonical substrate.
 21. The method of claim 20, wherein said non-canonical substrate comprises one or more alkene unsaturation sites.
 22. The method of claim 20, wherein the hydrogenase enzyme comprises a modified hydrophobic portion that supports the recognition of an unsaturated acyl chain.
 23. The method of claim 1, wherein said enzyme is a dehydrogenase enzyme engineered to bind a non-canonical substrate.
 24. The method of claim 23, wherein said non-canonical substrate comprises one or more alkene unsaturation sites.
 25. The method of claim 1, wherein said enzyme is a desaturase enzyme.
 26. The method of claim 25, wherein said desaturase enzyme is engineered.
 27. The method of claim 26, wherein said desaturase enzyme is engineered to bind a transition metal in its active site.
 28. The method of claim 27, wherein said active site comprises at least 2 cysteine residues that support transition metal binding.
 29. The method of claim 27, wherein the transition metal is nickel, iron, or palladium.
 30. The method of claim 27, wherein said active site comprises an arginine residue that is configured to support a frustrated Lewis pair reaction. 